CN102559648A - Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof - Google Patents
Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof Download PDFInfo
- Publication number
- CN102559648A CN102559648A CN2012100109857A CN201210010985A CN102559648A CN 102559648 A CN102559648 A CN 102559648A CN 2012100109857 A CN2012100109857 A CN 2012100109857A CN 201210010985 A CN201210010985 A CN 201210010985A CN 102559648 A CN102559648 A CN 102559648A
- Authority
- CN
- China
- Prior art keywords
- carrier
- enzyme
- epoxy
- immobilized enzyme
- ggt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000003822 epoxy resin Substances 0.000 title abstract 3
- 229920000647 polyepoxide Polymers 0.000 title abstract 3
- 102000004190 Enzymes Human genes 0.000 claims abstract description 72
- 108090000790 Enzymes Proteins 0.000 claims abstract description 72
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 claims abstract description 22
- 239000000872 buffer Substances 0.000 claims abstract description 21
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 claims abstract description 18
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 claims abstract description 18
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims abstract description 18
- 229940026510 theanine Drugs 0.000 claims abstract description 11
- 239000004593 Epoxy Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000012986 modification Methods 0.000 claims description 20
- 230000004048 modification Effects 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 230000003534 oscillatory effect Effects 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 208000028626 extracranial carotid artery aneurysm Diseases 0.000 description 56
- 102000018120 Recombinases Human genes 0.000 description 19
- 108010091086 Recombinases Proteins 0.000 description 19
- 238000000034 method Methods 0.000 description 10
- 239000011347 resin Substances 0.000 description 10
- 229920005989 resin Polymers 0.000 description 10
- 238000011084 recovery Methods 0.000 description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 5
- 230000003139 buffering effect Effects 0.000 description 5
- 239000012295 chemical reaction liquid Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 108010016626 Dipeptides Proteins 0.000 description 4
- 230000000274 adsorptive effect Effects 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 235000009508 confectionery Nutrition 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- -1 N, N '-methylene Chemical group 0.000 description 3
- 239000003708 ampul Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- LSWYGACWGAICNM-UHFFFAOYSA-N 2-(prop-2-enoxymethyl)oxirane Chemical compound C=CCOCC1CO1 LSWYGACWGAICNM-UHFFFAOYSA-N 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 150000002118 epoxides Chemical group 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- WMZTYIRRBCGARG-VIFPVBQESA-N (2s)-2-azaniumyl-5-(4-nitroanilino)-5-oxopentanoate Chemical compound OC(=O)[C@@H](N)CCC(=O)NC1=CC=C([N+]([O-])=O)C=C1 WMZTYIRRBCGARG-VIFPVBQESA-N 0.000 description 1
- JHPBZFOKBAGZBL-UHFFFAOYSA-N (3-hydroxy-2,2,4-trimethylpentyl) 2-methylprop-2-enoate Chemical compound CC(C)C(O)C(C)(C)COC(=O)C(C)=C JHPBZFOKBAGZBL-UHFFFAOYSA-N 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses an immobilized enzyme taking modified epoxy resin as a carrier, which comprises an enzyme and a modified carrier for fixing the enzyme, wherein the enzyme is gamma-glutamyl transpeptidase, and the modified carrier is epoxy resin Eupergit C modified by carrier ampholyte with the pH value of 8-10.5250L. The invention also discloses a preparation method and application of the immobilized enzyme. The immobilized enzyme obtained by the invention has the characteristics of uniform property, strong enzyme stability, good reusability, strong adaptability to the pH value of a reaction environment and the like, further improves the stability of the gamma-glutamyl transpeptidase, and realizes the application of the novel immobilized enzyme in theanine synthesis.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of immobilized enzyme.
Background technology
Biocatalytic reaction has characteristics such as mild condition, selectivity height, reaction efficiency height, thereby is applied to the field of chemical synthesis more and more.But, greatly limited the practical application of biocatalysis at industrial circle because the cost of poor stability and the zymin of enzyme under external industrial environment is too high.Enzyme immobilization technology also can significantly be improved the stability and the catalysis characteristics (substrate avidity, stereoselectivity etc.) of enzyme simultaneously through enzyme molecule constraint (or combination) in special phase, is not only efficiently solved the repeated use problem of catalyzer.
At present, as an important branch of enzyme immobilization technology, the development and application of high-performance enzyme immobilization carrier is very active.Research contents relates generally to synthetic, the sign of carrier, and the geometric parameter of carrier, surface properties, surfactivity and physico-chemical property etc. are for the influence of the aspects such as supported quantity, the enzyme work recovery and stability of enzyme.But relevant carrier surface charge property rarely has systematic research to the influence of enzymatic activity and stability.
Research shows, the surface charge property of the enzyme immobilization carrier of different substrates (pzc value) difference cause its surperficial environment PH to produce difference, and too high or too low pH value not only can reduce the catalytic efficiency (of enzyme, also will influence the stability of enzyme molecular structure simultaneously.Therefore, when selecting enzyme immobilization carrier, not only will consider the size of enzyme charge capacity and the stability that is connected, the while also need be taken into account the carrier surface charge property, and the righttest action condition of itself and enzyme is complementary.But, make that people's blindness when selecting carrier is bigger owing to lack the related data of all kinds of carrier surface charge properties at present.
Epoxy group(ing) carrier Eupergit C
250LBe a kind of enzyme immobilization carrier commonly used, by the porous small ball shape resin that monomer polymerization forms, participating in monomer polymerized has: N, N '-methylene radical-bisacrylamide, glycidyl allyl ether, methacrylic ester.Commercial Eupergit C
250LSize distribution is at the coccoid particle of 100～250 μ m, the micro-pore diameter 90～130nm on the resin, loading 200 μ mol epoxide group/g dried resins.Eupergit C
250LHave good chemicalstability, physical strength is high, and its pzc value is 6.54.At present with Eupergit C
250LFor the enzyme immobilization of carrier is studied a lot, but to the immobilization effect difference of various enzymes huge (the enzyme recovery alive does not wait from 8%～92%).Although do not get rid of that immobilization process stretches to the mechanicalness of enzyme conformation and distortion and to the influence of mass transfer, carrier self charge environment also is huge to the influence of enzyme texture image and apparent catalytic performance.At present, through at Eupergit C
250LThe carrier ampholyte of the specific pH buffering range of surface grafting, preparation has the functional carrier of shock-absorbing capacity, and the research of using it for enzyme immobilization does not appear in the newspapers as yet.
Gamma glutamyl transpeptidase (γ-glutamyltranspeptidase; GGT; The righttest action pH 8.5～9.5) can specifically γ-Gu Anxianji be transferred to acceptor molecule, obtain the new compound that contains γ-Gu Anxianji, this reaction becomes the glutamyl reaction.Can obtain different glutamy compounds through changing acceptor specy.Since this reaction have locus specificity and optical selective strong, need not to reactant protect and deprotection, reaction process in do not consume characteristics such as ATP yet, therefore utilize the research of the serial γ-Gu Anxianji compounds of this prepared in reaction becoming the research focus of industrial biocatalysis field.This reaction at present successfully is applied to the synthetic of some important glutamy compounds, like γ-L-glutamy-L-DOPA, gsh, gamma-D-glutamine acyl L-tryptophane, gamma-L-glutamine-cysteine and theanine etc.But because GGT is prone to the subunit depolymerization takes place, cause the irreversible decline that enzyme is lived under external environment, limited the further expansion of its range of application.
At present, less to the immobilization and the stabilization research of gamma glutamyl transpeptidase, and material modifiedly with carrier ampholyte be the GGT immobilization of carrier and use and all not appear in the newspapers.
Summary of the invention
It is the immobilized enzyme of carrier with the epoxy after the modification that technical problem to be solved by this invention provides a kind of; With the flexibility of further raising gamma glutamyl transpeptidase to environment pH value; And the stability of enzyme and catalytic activity, reduce the technology cost for preparing theanine.
The technical problem that the present invention also will solve provides the preparation method of said fixing enzyme.
The technical problem that the present invention will solve at last provides the application of said fixing enzyme in the synthetic theanine of catalysis.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of epoxy with modification is the immobilized enzyme of carrier; It comprises the modified support of enzyme and fixing said enzyme; Described enzyme is a gamma glutamyl transpeptidase, and described modified support is through the epoxy Eupergit C after the carrier ampholyte modification of pH8～10.5
250L
Wherein, described modified support prepares as follows: with dried epoxy Eupergit C
250LAdd in the carrier ampholyte of pH8～10.5; Under the normal temperature behind stirring reaction 16～20h; Liquid is leached, solid particulate respectively through the NaCl solution drip washing repeatedly of pure water drip washing repeatedly, 0.5mol/L, pure water repeatedly after the drip washing three road drip washing oven dry promptly make modified support.
Wherein, the carrier ampholyte of described pH8～10.5 is pharmalyte
TM8-10.5, liquid state, CAS number is 17-0455-01, and buffering range is pH 8～10.5, and production firm is GE healthcare, and original solution concentration is 0.36meq/mL.
Wherein, described epoxy Eupergit C
250LFor
C250L, CAS number is 149531-08-8, production firm is FLUKA, Eupergit
TMC
250LBe N; N '-methylene radical-bisacrylamide, glycidyl allyl ether, alkylmethacrylate polymer; Size distribution is at the coccoid particle of 100～250 μ m, the micro-pore diameter 90～130nm on the resin, and epoxide group concentration is more than or equal to 200 μ mol/g dried resins.
Wherein, for every g dry weight epoxy Eupergit C
250L, the consumption of carrier ampholyte is 10～20mL.
Wherein, the carrier ampholyte use concentration with pH8～10.5 is the carrier ampholyte aqueous solution replacement of above pH8～10.5 of 50% (v/v).
Wherein, described drying condition is vacuum drying oven inner drying 48～72h, and drying temperature is not higher than 40 ℃.
Above-mentioned epoxy with modification is the preparation method of the immobilized enzyme of carrier; Modified support is added in the gamma glutamyl transpeptidase solution, and oscillatory reaction 1～5h under normal temperature (preferred 2.5h) leaches liquid; Solid particulate pure water drip washing 3～4 times promptly gets after the oven dry.
Wherein, the concentration of described gamma glutamyl transpeptidase solution is 0.25～0.30mg/mL.
Wherein, for every g dry weight modified support, the consumption of gamma glutamyl transpeptidase solution is 10～20mL.
Wherein, described drying condition is vacuum drying oven inner drying 48～72h, and drying temperature is not higher than 40 ℃.
The final water wash of immobilized enzyme that the above-mentioned epoxy with modification for preparing is a carrier dries up and is placed on 4 ℃ of preservations of refrigerator, and the immobilized enzyme that makes has good chemical stability, Stability Analysis of Structures, has good power operation intensity, surface to be rich in-NH
2With-COOH, very easily with intermolecular formation hydrogen bond of enzyme or ionic linkage.
The application of the immobilized enzyme that above-mentioned epoxy with modification is a carrier in the synthetic theanine of catalysis.
Carrier ampholyte: Pharmalyte is hereinafter to be referred as CA.
Through the epoxy Eupergit C after the carrier ampholyte modification of pH8～10.5
250L, i.e. modified support is hereinafter to be referred as ECCA.
Epoxy with above-mentioned modification is the immobilization gamma glutamyl transpeptidase of carrier, hereinafter to be referred as ECCA-GGT.
Beneficial effect: the present invention makes full use of epoxy Eupergit C
250LSuperperformance; Through preparing novel vector ECCA at the carrier ampholyte of its surperficial covalent bonding buffering range in pH8～10.5; The ECCA zero point charge value for preparing is 9.1; Near the righttest action pH value of GGT, and ECCA support shapes homogeneous, stability are high, be rich on the surface-COOH and-NH
2, for the immobilization gamma glutamyl transpeptidase provides good operational condition.
With the ECCA-GGT shape homogeneous of the inventive method preparation, enzymatic activity recovery on average reaches 19.22%; In pure water, the maximum reaction velocity of immobilized enzyme ECCA-GGT is 81%～95.8% under the buffer system; Compare with resolvase, the righttest action pH and the temperature of immobilized enzyme slightly change, and applicable pH and TR all have increase to a certain degree; The thermostability of immobilized enzyme and pH stability all are improved largely than resolvase, and repeat performance is good.
Description of drawings
Fig. 1 is the absorption-time curve of ECCA to GGT.Because ECCA is through physical adsorption to the immobilized of GGT, its adsorptive capacity is relevant with the specific surface area of carrier, and can form multilayer adsorption.Therefore, carrier can reach 2.7mg/g resin and 19.29% to the equilibrium adsorption capacity and the enzyme recovery alive of enzyme, than Eupergit C
250LRaising is by a relatively large margin arranged.
Fig. 2 is the influence of temperature to ECCA-GGT immobilized enzyme and resolvase vigor.This schemes to show that the optimal reactive temperature of immobilized enzyme ECCA-GGT and free GGT is all about 60 ℃.But ECCA-GGT decreases to the susceptibility of temperature, and under room temperature (23.7 ℃) condition, the relative enzyme work of ECCA-GGT still can reach more than 60% of the highest enzyme work.
Fig. 3 is the influence of pH value to immobilized enzyme and resolvase vigor.This schemes to show that the righttest action pH value of resolvase and immobilized enzyme is all about 9.0.But immobilized enzyme obviously descends in the susceptibility that the pH value is changed, and when the pH value was 6.0, the relative enzyme of immobilized enzyme was lived still up to about 70%, only remains 40.44% and the relative enzyme of resolvase is alive at this moment.
Fig. 4 be the ECCA-GGT immobilized enzyme pure water (A) and Tris-HCl buffer system (pH 8.0) (B) the kinetic constant fitting result.This schemes to show the maximum reaction velocity (r of immobilized enzyme in pure water
Max) be 0.0236mmol/L.min, for its in the Tris-HCl damping fluid 116.5%.
Fig. 5 is that the thermostability of resolvase and ECCA-GGT immobilized enzyme compares.This schemes to show that the thermostability of ECCA-GGT is greatly improved than resolvase.
Fig. 6 is the operational stability of ECCA-GGT.This schemes to show that the operational stability of ECCA-GGT is good.Through 19 batches repeated use, the relative enzyme of immobilized enzyme ECCA-GGT is lived and still can be reached more than 50% of initial value.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The enzyme activity detection method is following in following examples:
Enzyme is lived and defined: with gamma-glutamyl p-Nitroaniline (GpNA) is donor, is acceptor with two sweet dipeptides, and PM catalysis GpNA hydrolysis generates the required enzyme amount of 1umol p-Nitroaniline (pNA)
P-Nitroaniline (pNA) typical curve: the preparation different concns (1～5mmol/L) pNA standardized solution, and under 410nm, measure its light absorption value, be horizontal, vertical coordinate with pNA concentration with corresponding light absorption value respectively, the drawing standard curve.
The resolvase enzyme activity determination: get enzyme liquid 50 μ l, add Tris-HCl, constant volume is put 10mL (200 times of enzyme liquid dilutions).Get dilution back enzyme liquid 0.5mL, add 0.5mL GpNA (concentration 5mmol/L), the two sweet dipeptides of 0.5mL, 1.5mLTris-HCl buffer, total reaction system 3.0mL.37 ℃ of water-bath 5～15min.Reaction solution is in A
410Measure light absorption value.Immobilized enzyme enzyme activity determination: get immobilized enzyme 10～30mg, add 0.5mL GpNA, the two sweet dipeptides of 0.5mL, 2.0mLTris-HCl buffer, total reaction system 3.0mL.37 ℃ of water-bath 5～10min.Reaction solution leaches the back in A
410Measure light absorption value.
Enzyme recovery method of calculation alive are following in following examples:
The recovery=(immobilized enzyme vigor/always drop into enzyme to live) * 100%
In following examples, Eupergit C
250LAvailable from FLUKA company, carrier ampholyte pharmalyte
TM8--10.5 available from GE Healthcare company, gamma glutamyl transpeptidase is self-control, gained GGT is that electrophoresis is pure, and enzyme is lived and is 8.12U/mL, and protein concentration is 0.5mg/mL.
Embodiment 1:ECCA preparing carriers.
Get the Eupergit C of 0.3g oven dry
250LPlace in the 20ml core pipe, according to the CA aqueous solution of solid-to-liquid ratio 1g/20mL adding 50% (v/v), normal-temperature reaction 16h will leach reaction liquid earlier after reaction finishes.Solid particulate after filter is done is with pure water drip washing 6～7 times repeatedly; The drip washing 3～4 times repeatedly of the NaCl solution of using 0.5mol/L again; Do with filter after the pure water drip washing 6～7 times more at last, promptly make the carrier ECCA that CA modifies in 40 ℃ of oven dry.Grafting amount by subtraction CA of poor quality is 0.0964g.
Embodiment 2:ECCA preparing carriers.
Take by weighing the Eupergit C of 0.3g oven dry
250LPlace in the 20ml core pipe, add 100% (v/v) CA according to solid-to-liquid ratio 1g/10mL, 24 ℃ of room temperature reaction 20h will leach reaction liquid earlier after reaction finishes.Solid particulate after filter is done is with pure water drip washing 6～7 times repeatedly; The drip washing 3～4 times repeatedly of the NaCl solution of using 0.5mol/L again; Do with filter after the pure water drip washing 6～7 times more at last, promptly make the carrier ECCA that CA modifies in 40 ℃ of oven dry.Grafting amount by subtraction CA of poor quality is 0.124g.
The preparation method of embodiment 3:ECCA-GGT.
Take by weighing the dried finished product carrier ECCA 0.3g for preparing with most preferred method among the embodiment 2 as in the ampoule; By the GGT enzyme liquid (enzyme concn is 0.3mg/mL) behind solid-to-liquid ratio 1g: the 13.3mL adding purifying; Bathe shaking table oscillatory reaction 1h in water at normal temperature; Reaction finishes the back and earlier reaction liquid is leached, and the solid particulate after filter is done promptly makes ECCA-GGT with pure water drip washing 3～4 times repeatedly in 30 ℃ of oven dry.The GGT adsorptive capacity is the 1.26mg/g resin, and the enzyme recovery alive is 9.72%.
The preparation method of embodiment 4:ECCA-GGT.
Take by weighing the dried finished product carrier ECCA 3.0g for preparing with most preferred method among the embodiment 2 as in the ampoule; By the GGT enzyme liquid (enzyme concn is 0.25mg/mL) behind solid-to-liquid ratio 1g: the 20mL adding purifying; Bathe shaking table oscillatory reaction 2.5h in water at normal temperature; Reaction finishes the back and earlier reaction liquid is leached, and the solid particulate after filter is done promptly makes ECCA-GGT with pure water drip washing 3～4 times repeatedly in 30 ℃ of oven dry.The GGT adsorptive capacity is the 2.7mg/g resin, and the enzyme recovery alive is 19.29%.
The preparation method of embodiment 5:ECCA-GGT.
Take by weighing the dried finished product carrier ECCA 3.0g for preparing with most preferred method among the embodiment 2 as in the ampoule; By the GGT enzyme liquid (enzyme concn is 0.3mg/mL) behind solid-to-liquid ratio 1g: the 10mL adding purifying; Bathe shaking table oscillatory reaction 5h in water at normal temperature; Reaction finishes the back and earlier reaction liquid is leached, and the solid particulate after filter is done promptly makes ECCA-GGT with pure water drip washing 3～4 times repeatedly in 30 ℃ of oven dry.The GGT adsorptive capacity is the 2.68mg/g resin, and the enzyme recovery alive is 19.21%.
Embodiment 6:ECCA-GGT catalytic preparation theanine.
Theanine (Theanine) belongs to amides, and systematic naming method is N-ethyl-γ-L-glutaminate (N-ethyl-γ-L-glutamine).With the gamma glutamyl transpeptidase is catalyzer, and L-glutaminate (Gln) and ethamine are substrate, can realize that enzyme process synthesizes theanine.Preparation Gln and ethamine concentration are all 5mmol/L, add resolvase GGT, and the immobilized enzyme ECCA-GGT that presses the preparation of embodiment 4 methods, and in 40 ℃ of water-baths, timing sampling is measured the concentration of product theanine.Experimental result shows, is catalyzer with immobilized enzyme of the present invention, and under identical enzyme concn, through reaction 24h, the donor transformation efficiency is 62.3%; When being catalyzer with the resolvase, the donor transformation efficiency is merely 34.1%.This reaction system is simple, and mild condition can realize operate continuously.
Embodiment 7: the shock-absorbing capacity comparison test: the kinetic constant of ECCA-GGT immobilized enzyme under pure water and Tris-HCl buffer system measured.
Optimal fixed condition according to above-mentioned prepares ECCA-GGT, in pure water and Tris-HCl buffering (pH8.0), measures the kinetic constant of immobilized enzyme respectively.The concentration of the two sweet dipeptides of sessile receptor is 100mmol/L in the experiment, and donor GpNA concentration is 0.5～5mmol/L, measures the GGT speed of reaction under the different GpNA concentration.Through the match of single substrate Michaelis-Menton equation, obtain the maximum response speed of immobilized enzyme ECCA-GGT under pure water and Tris-HCl buffer system and be respectively 0.0275mmol/L.min and 0.0236mmol/L.min (as shown in Figure 4).The maximum response speed of ECCA-GGT in pure water is 116.5% under the buffer system in the pure water.
Embodiment 8: the optimum temperuture of resolvase and ECCA-GGT immobilized enzyme relatively.
Optimal fixed condition according to above-mentioned prepares ECCA-GGT; Under differing temps (23.7～70 ℃), measure resolvase and immobilized enzyme vigor respectively, the result is as shown in Figure 2: the optimal reactive temperature of immobilized enzyme ECCA-GGT and free GGT is all about 60 ℃.But ECCA-GGT decreases to the susceptibility of temperature, and under room temperature (23.7 ℃) condition, the relative enzyme work of ECCA-GGT still can reach more than 60% of the highest enzyme work.
Embodiment 9: resolvase, Eupergit C
250LThe optimum pH of-GGT and ECCA-GGT relatively.
Optimal fixed condition according to above-mentioned prepares ECCA-GGT.By the method for Song Xiwen etc. (see " modern chemical industry " for details, 2009,29 (supplementary issues 2): 147～149), with without the Eupergit C that modifies
250LBe carrier, add the GGT behind the purifying by solid-to-liquid ratio 1: 10, mixture is in 4 ℃ of following oscillatory reaction 24h, behind the filtering raffinate, after repeatedly washing with 10 times of Tris-HCl damping fluids to amount of resin (v/w) (50mmol/L, pH 8.0), makes immobilized enzyme Eupergit C
250L-GGT.
In the solution of pH 7～9 (Tris-HCl buffering) and pH10～11 (sodium bicarbonate buffer), measure resolvase, Eupergit C respectively
250LThe vigor of-GGT and ECCA-GGT, the result is as shown in Figure 3: the righttest action pH value of resolvase and immobilized enzyme is all about 9.0.But ECCA-GGT obviously descends to the susceptibility that the pH value changes, and when pH value was 6.0, the relative enzyme of ECCA-GG was lived still up to about 70%, and resolvase and Eupergit C at this moment
250LThe relative enzyme of-GGT is lived and is only remained 40.44% and 17.9%.Show the Eupergit C that modifies with CA
250LFor carrier carries out the action pH scope that immobilization can enlarge enzyme effectively to GGT.
The experiment of embodiment 10:ECCA-GGT immobilized enzyme thermostability.
Get a certain amount of free GGT and ECCA-GGT (30～50 ℃) insulation under differing temps respectively, sampling and measuring GGT enzyme is lived at regular intervals, is 100% with 0 constantly GGT activity, calculates the relative enzyme Ar that lives.Experimental result shows that the thermostability of ECCA-GGT has rising (Fig. 5 A and 5B) by a relatively large margin than resolvase.Through match, the heat inactivation reaction activity of ECCA-GGT is E
d=125.4KJ/mol is than resolvase (E
d=49.8KJ/mol) be improved largely.
The test of embodiment 11:ECCA-GGT stability in use.
Take by weighing the ECCA-GGT of 18.1mg, in 4 ℃ of storages down, the results of regular determination enzyme is lived, the stable storing property testing of immobilized enzyme, and the result is as shown in Figure 6: the operational stability of ECCA-GGT is good.Through 19 batches repeated use, the relative enzyme of immobilized enzyme ECCA-GGT is lived and still can be reached more than 50% of initial value.
Claims (10)
1. the epoxy with modification is the immobilized enzyme of carrier; It is characterized in that; It comprises the modified support of enzyme and fixing said enzyme; Described enzyme is a gamma glutamyl transpeptidase, and described modified support is through the epoxy Eupergit C after the carrier ampholyte modification of pH8～10.5
250L
2. the epoxy with modification according to claim 1 is the immobilized enzyme of carrier, it is characterized in that described modified support prepares as follows: with dried epoxy Eupergit C
250LAdd in the carrier ampholyte of pH8～10.5, behind stirring reaction 16～20h, liquid leached under the normal temperature, solid particulate respectively through the NaCl of pure water, 0.5mol/L solution and pure water repeatedly the drip washing after drying promptly make modified support.
3. the epoxy with modification according to claim 4 is the immobilized enzyme of carrier, it is characterized in that, for every g dry weight epoxy Eupergit C
250L, the consumption of carrier ampholyte is 10～20mL.
4. the epoxy with modification according to claim 4 is the immobilized enzyme of carrier, it is characterized in that, it is the carrier ampholyte aqueous solution replacement of above pH8～10.5 of 50% (v/v) that the carrier ampholyte of pH8～10.5 is used concentration.
5. the epoxy with modification according to claim 4 is the immobilized enzyme of carrier, it is characterized in that, described drying condition is vacuum drying oven inner drying 48～72h, and drying temperature is not higher than 40 ℃.
6. the described epoxy with modification of claim 1 is the preparation method of the immobilized enzyme of carrier, it is characterized in that, modified support is added in the gamma glutamyl transpeptidase solution; Oscillatory reaction 1～5h under normal temperature; Liquid is leached, and solid particulate pure water drip washing 3～4 times promptly gets after the oven dry.
7. the epoxy with modification according to claim 7 is the preparation method of the immobilized enzyme of carrier, it is characterized in that, the concentration of described gamma glutamyl transpeptidase solution is 0.25～0.30mg/mL.
8. the epoxy with modification according to claim 7 is the preparation method of the immobilized enzyme of carrier, it is characterized in that, for every g dry weight modified support, the consumption of gamma glutamyl transpeptidase solution is 10～20mL.
9. the epoxy with modification according to claim 7 is the preparation method of the immobilized enzyme of carrier, it is characterized in that, described drying condition is vacuum drying oven inner drying 48～72h, and drying temperature is not higher than 40 ℃.
10. the described epoxy of claim 1 application of immobilized enzyme in the synthetic theanine of catalysis that be carrier with modification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210010985.7A CN102559648B (en) | 2012-01-13 | 2012-01-13 | Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210010985.7A CN102559648B (en) | 2012-01-13 | 2012-01-13 | Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559648A true CN102559648A (en) | 2012-07-11 |
| CN102559648B CN102559648B (en) | 2014-04-02 |
Family
ID=46406255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210010985.7A Expired - Fee Related CN102559648B (en) | 2012-01-13 | 2012-01-13 | Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559648B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851272A (en) * | 2012-09-25 | 2013-01-02 | 南京工业大学 | Gamma-glutamyltranspeptidase immobilized enzyme and preparation method and application thereof |
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN103937776A (en) * | 2014-04-10 | 2014-07-23 | 南京工业大学 | Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by carrier ampholyte |
| CN106222157A (en) * | 2016-08-12 | 2016-12-14 | 南京工业大学 | Immobilized enzyme with polyamino acid modified polystyrene resin as carrier and preparation method thereof |
| CN110964712A (en) * | 2019-12-16 | 2020-04-07 | 牡丹江医学院 | Preparation method of immobilized enzyme for synthesizing gamma-L-glutamyl-L-cysteine |
| CN112679701A (en) * | 2020-12-28 | 2021-04-20 | 重庆宸安生物制药有限公司 | Immobilized lysine endopeptidase and preparation method and application thereof |
| CN114606221A (en) * | 2022-05-12 | 2022-06-10 | 凯莱英医药集团（天津）股份有限公司 | Immobilized enzyme, preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526474A (en) * | 2003-09-22 | 2004-09-08 | 南京工业大学 | Method for synthesizing titanium oxide with high specific surface area |
| CN101775387A (en) * | 2010-03-23 | 2010-07-14 | 南京工业大学 | Mesoporous titanium dioxide immobilized enzyme and preparation method and application thereof |
-
2012
- 2012-01-13 CN CN201210010985.7A patent/CN102559648B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526474A (en) * | 2003-09-22 | 2004-09-08 | 南京工业大学 | Method for synthesizing titanium oxide with high specific surface area |
| CN101775387A (en) * | 2010-03-23 | 2010-07-14 | 南京工业大学 | Mesoporous titanium dioxide immobilized enzyme and preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| 《化学反应工程与工艺》 20091031 宋希文等 固定化gamma-谷氨酰转肽酶催化合成gamma-L-谷氨酰-L-半胱氨酸 420－425 1－10 第25卷, 第5期 * |
| 《现代化工》 20091031 肖彦羚等 固定化gamma-谷氨酰转肽酶的催化特性研究 147－149 1－10 第29卷, * |
| WANG T ET AL: "On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis", 《J CHROMATOGR B ANALYT TECHNOL BIOMED LIFE SCI.》 * |
| YANJUN JIANG ET AL.: "The improved stability of enzyme encapsulated in biomimetic titania particles", 《MATERIALS SCIENCE AND ENGINEERING C》 * |
| 宋希文等: "固定化γ-谷氨酰转肽酶催化合成γ-L-谷氨酰-L-半胱氨酸", 《化学反应工程与工艺》 * |
| 肖彦羚等: "固定化γ-谷氨酰转肽酶的催化特性研究", 《现代化工》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851272A (en) * | 2012-09-25 | 2013-01-02 | 南京工业大学 | Gamma-glutamyltranspeptidase immobilized enzyme and preparation method and application thereof |
| CN102851272B (en) * | 2012-09-25 | 2013-10-09 | 南京工业大学 | Gamma-glutamyltranspeptidase immobilized enzyme and preparation method and application thereof |
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN103864967B (en) * | 2012-12-11 | 2016-03-23 | 中国科学院大连化学物理研究所 | Pharmalyte modify polymer beads and apply in protein example pre-treatment |
| CN103937776A (en) * | 2014-04-10 | 2014-07-23 | 南京工业大学 | Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by carrier ampholyte |
| CN106222157A (en) * | 2016-08-12 | 2016-12-14 | 南京工业大学 | Immobilized enzyme with polyamino acid modified polystyrene resin as carrier and preparation method thereof |
| CN106222157B (en) * | 2016-08-12 | 2019-02-22 | 南京工业大学 | Immobilized enzyme with polyamino acid modified polystyrene resin as carrier and preparation method thereof |
| CN110964712A (en) * | 2019-12-16 | 2020-04-07 | 牡丹江医学院 | Preparation method of immobilized enzyme for synthesizing gamma-L-glutamyl-L-cysteine |
| CN112679701A (en) * | 2020-12-28 | 2021-04-20 | 重庆宸安生物制药有限公司 | Immobilized lysine endopeptidase and preparation method and application thereof |
| CN114606221A (en) * | 2022-05-12 | 2022-06-10 | 凯莱英医药集团（天津）股份有限公司 | Immobilized enzyme, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559648B (en) | 2014-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102559648B (en) | Immobilized enzyme with modified epoxy resin as carrier and preparation method and application thereof | |
| Ren et al. | Carbonic anhydrase@ ZIF-8 hydrogel composite membrane with improved recycling and stability for efficient CO2 capture | |
| Brena et al. | Immobilization of enzymes: a literature survey | |
| Tüzmen et al. | Immobilization of catalase via adsorption onto metal-chelated affinity cryogels | |
| Cantone et al. | Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods | |
| Górecka et al. | Immobilization techniques and biopolymer carriers | |
| Sarı et al. | Reversible immobilization of catalase by metal chelate affinity interaction on magnetic beads | |
| Wang et al. | Immobilization of cellulase on polyamidoamine dendrimer-grafted silica | |
| Srivastava et al. | Boronate affinity chromatography of cells and biomacromolecules using cryogel matrices | |
| CN102068965B (en) | Method for preparing chitosan separation medium suitable for protein purification | |
| Corona-González et al. | Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid | |
| Uygun et al. | Novel magnetic nanoparticles for the hydrolysis of starch with Bacillus licheniformis α‐amylase | |
| Yu et al. | Immobilization of glucose isomerase onto GAMM support for isomerization of glucose to fructose | |
| Bai et al. | Study on synthesis of a hydrophilic bead carrier containing epoxy groups and its properties for glucoamylase immobilization | |
| Jiang et al. | Facile immobilization of enzyme on three dimensionally ordered macroporous silica via a biomimetic coating | |
| Acet et al. | Determination of some adsorption and kinetic parameters of α-amylase onto Cu+ 2-PHEMA beads embedded column | |
| Li et al. | Nano-sized mesoporous hydrogen-bonded organic frameworks for in situ enzyme immobilization | |
| Akgöl et al. | Reversible immobilization of urease onto Procion Brown MX-5BR-Ni (II) attached polyamide hollow-fibre membranes | |
| WO2015081879A1 (en) | Sn-1,3 selective immobilized lipase for catalytic esterification and ester interchange and method for preparing same | |
| Bayramoğlu et al. | Immobilization of urease via adsorption onto l-histidine–Ni (II) complexed poly (HEMA-MAH) microspheres: preparation and characterization | |
| Li et al. | Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review | |
| Xi et al. | Preparation of macroporous chitosan layer coated on silica gel and its application to affinity chromatography for trypsin inhibitor purification | |
| Mao et al. | Flow‐through enzymatic reactors using polymer monoliths: from motivation to application | |
| Xu et al. | Robust enhancing stability and fructose tolerance of sucrose phosphorylase by immobilization on Ni-NTA functionalized agarose microspheres for the biosynthesis of 2-α-glucosylglycerol | |
| Tan et al. | Lipase-polydopamine magnetic hydrogel microspheres for the synthesis of octenyl succinic anhydride starch |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140402 Termination date: 20150113 |
|
| EXPY | Termination of patent right or utility model |